Adeno-Associated Virus (AAV) gene therapy vectors are produced using producer cells grown in culture. Producer cells either release the AAV particles into the cell culture media or they remain associated with the cell, wherein the ratio released to retained AAV particles depends, in part, on the “serotype” of the AAV. Current methods of releasing cell-associated AAV are often time consuming, difficult to scale, or can introduce unwanted and difficult to remove impurities such as detergents.
Various methods have been proposed to improve and/or simplify the recovery of virus or viral products from feedstock. U.S. Pat. No. 3,316,153 describes a multi-step extraction process, aimed at separating virus particles from cellular debris and is assertedly applicable to feedstocks derived from virus-infected chick allantoic fluid or from cell or tissue-culture fluids. In this method, virus adsorbed to precipitated calcium phosphate is dispersed in EDTA at pH 7.8-8.3, causing dissociation and an EDTA-based sequestering of the soluble calcium, thereby releasing the virus for recovery. The resulting virus-containing solution is dialyzed against water or preferably an aqueous glycine-sodium chloride solution to reduce the EDTA and phosphate content.
In U.S. Pat. No. 3,874,999, allantoic fluids containing influenza virus are centrifuged at low speeds to remove gross particles. The virus is then removed from the supernatant by high-speed centrifugation and resuspended in a phosphate buffer. Nonvirus proteins and lipids are removed by treatment of the suspension with 0.1-0.4 M magnesium sulfate at an alkaline pH for 16-18 hours at 4° C. The resultant suspension is clarified by low speed centrifugation and the virus is purified from the resulting supernatant.
U.S. Pat. No. 3,962,421 describes a method for the disruption of influenza viruses. Allantoic fluid is subjected to high-speed centrifugation. The resulting pellet is resuspended in saline and ball-milled for 12-15 hours to create a virus suspension. The virus suspension is then treated with phosphate-ester to disrupt the virus particles into lipid-free particles (subunits) that carry the surface antigens of intact viruses.
In U.S. Pat. No. 4,327,182, allantoic fluid feedstocks from the growth of influenza virus are subjected to a multi-stage extraction process aimed at recovering influenza subunits, haemagglutinin and neuraminidase. The technique relies on a concentration step in which virus feedstock is present with detergent and a saline solution followed by successive filtration to remove non-viral particles.
U.S. Pat. No. 4,724,210 describes methods for purification of influenza using ion exchange chromatography. An influenza-containing solution, e.g., allantoic fluid, is passed through cellulose sulfate column wherein the virus is adhered to the column packing. The column is subsequently washed and virus eluted with a solution containing 1.0 M to 1.5 M sodium chloride. This is followed by a 4.99 M sodium chloride wash.
U.S. Pat. No. 6,566,118 discloses a method of generating a population of recombinant AAV (rAAV) particles by incubating producer cells in a cell culture medium under conditions that promote release of rAAV particles into the medium without lysing the cells. Culture conditions of use in promoting release of rAAV include pH, osmolality (e.g., by using an ionic salt), and temperature.
In WO 1999/07834, herpesvirus infected Vero cell cultures are incubated in a hypertonic aqueous salt solution (0.8 to 0.9 M NaCl) for several hours. The solution is then removed and herpesvirus harvested from the solution. This method is asserted to be superior to methods wherein the cells are subjected to ultrasonic disruption.
US 2005/0186223 discloses a method for dissociating virus from virus-debris complexes by increasing the salt concentration of allantoic fluid. This reference teaches that the salt can be monovalent, divalent or multivalent cation mixtures thereof, e.g., NaCl, KCl, LiCl, CaCl2, MgCl2 and other salts, and can include or exclude ammonium sulfate.
US 2005/0266567 describes methods for generating high titer helper-free preparations of released rAAV vectors using osmolality to release the rAAV from producer cells.
US 2015/0024467 discloses methods of isolating a population of rAAV from in-process impurities by capturing the rAAV particles on an apatite chromatography medium in the presence of polyethylene glycol (PEG). The methods can entail upstream processing such as centrifugation, treatment with BENZONASE®, anion exchange filtration, and/or tangential flow filtration, as well as downstream processing such as heat inactivation, filtration, hydrophobic interaction chromatography, size exclusion chromatography, and/or anion exchange chromatography.
In WO 2002/067983, preparation of a split influenza vaccine is described as involving moderate-speed centrifugation to clarify allantoic fluid, adsorption of the clarified fluid on a CaHPO4 gel, followed by resolublization with an EDTA-Na2 solution. See also WO 02/08749 describing the same process.
During et al. ((1998) Gene Therapy 5:820-827) describe the use of a combination of salts (i.e., NaCl, KCl, CaCl2 and MgCl2) to release rAAV from HEK 293 cells.
In Lock, et al. (2010) Human Gene Therapy 21(10): 1259-1271), the release of AAV serotypes into culture medium was investigated by the addition of 500 mM NaCl.
Ammonium sulfate has also been suggested for use in concentrating AAV by precipitation (Piras, et al. (2013) PLoS One 8:1-11; Piras, et al. (2016) Gene Ther. 23(5):469-78). Further, hydrophobic interaction chromatography (HIC) has been suggested for use in the isolation of AAV (Chalal, et al. (2007) J. Virol. Methods 139:61-70).
Methods for improving yields by contacting virus-infected cultured cells with elevated salt concentrations have also been described. For example, U.S. Pat. No. 5,506,129 reports increased yields of hepatitis A virus after growing infected BS-C-1 cells in growth medium containing 0.3 M NaCl.
Further, modulation of viral characteristics has been assessed by contacting purified viruses with elevated salt concentrations. In Breschkin et al. ((1977) Virology 80:441-444), a particular mutated measles virus lacking hemagglutination activity in isotonic saline exhibited wild-type level hemagglutination activity in 0.8 M (NH4)2SO4, whereas the high salt had no effect on the hemagglutination activity of a wild-type virus.
Wallis & Melnick ((1962) Virology 16:504-506) reported that, while high salt (1 M MgCl2, 1 M CaCl2, or 2 M NaCl) prevents heat inactivation of polio, coxsackie, and ECHO viruses, 1 M MgCl2 enhances inactivation of adeno-, papova-, herpes-, myxo-, arbor, and poxviruses.
In U.S. Pat. No. 7,704,721, aggregation of purified rAAV virions was prevented by adding at least 200 mM citrate, phosphate, sulfate or magnesium salt to the purified rAAV virions.